Fluorescent derivatization of a protease antigen to track antigen uptake and processing in human cell lines

Background We have devised a simple and efficient fluorescence-based method to track antigen uptake and processing in human B lymphoblastoid cells (B-LCL). Fluorescein labelled subtilisin was used to optimize antigen uptake conditions and identify processed peptides from human cell lines. Results Fluorescein labelled subtilisin conjugates had 0.06 to 2 moles of fluorescein per subtilisin molecule. High performance liquid chromatography and mass spectrometry (NanoESI-LC/MS/MS) analysis identified fluorescein conjugated to K141, K256, and the N terminus. Conjugates retained antigenic specificity to subtilisin specific antibodies and could be processed by whole cell extracts into low molecular weight fragments at pH 5.2. Maximal antigen uptake and processing occurred when PMSF (phenylmethylsulfonyl fluoride) inhibited subtilisin conjugate was incubated with cells at 100–200 μg/ml for 16 to 24 hr. Once optimal uptake conditions were established, processed subtilisin peptides were isolated and identified from human cell lines. Conclusion Our studies show that FITC-conjugation provides an efficient tool to track the uptake and processing of this protease antigen and to facilitate identification of processed antigenic peptides from human cell lines.


Background
Antigen presentation by MHC class I and class II molecules is a key aspect of an effective immune response against infections, cancer, certain allergic responses and autoimmune disease. Several good reviews cover capture and processing of exogenous antigens [1,2]. The detection and identification of antigen-derived peptides is important to understand the mechanisms involved in the immune response and to be able to modulate it. Though some studies have employed synthetic peptides to detect peptide epitopes, this approach is unable to identify naturally processed and presented peptides. Some of the early methods employed for identification of naturally processed endogenous epitopes were HPLC and Edman degra-dation, which led to the identification of hen egg lysozyme (HEL) 52-61 on the MHC of murine B lymphoma cells expressing HEL [3]. Hunt et al. showed the power of joining immunological procedures and mass spectrometry to identify self-peptides presented on cell surfaces in picomole or less concentrations from mouse and human cell lines [4][5][6]. Using mass spectrometry, Vignali et al. identified 16 peptides from HEL, which contained the minimal HEL 52-61 epitope as the single immunodominant epitope in a nested set of peptides [4]. Using tandem mass spectrometry and functional analysis with SEQUEST software, Dongre, et al. identified 128 selfpeptide sequences displayed by murine MHC class II molecules [7].
In the case of exogenous antigens such as ovalbumin [8], tetanus toxoid [9], Goodpasture antigen [10] and diabetes autoantigen [11], many investigators have used a modified delivery system to identify class II MHC peptides. Studies on exogenous class II peptides are limited as a result of requiring large amounts of purified antigen with over 10 9 cells, and the target peptides appearing in picomole quantities or less [12], often present as a subset of nested peptides thus diluting the mass spectrometric signal and rendering detection difficult.
Previous studies from our laboratory have identified class II epitopes from the subtilisin antigen, using synthetic subtilisin peptides with human dendritic cells and naive CD4+ T cells derived from the same donor [13]. In order to identify naturally processed epitopes, we have developed an efficient method of tracking protein in the cell using fluorescein-conjugated antigen. Fluorescent proteins have been used to track microvascular permeability [14], to study intestinal epithelial cell uptake [15], to study substrate processing [16,17] and to track proteins on cells with flow cytometry [18,19]. With the availability of the latest mass spectrometric methods, we investigated the use of fluorescein to study antigen uptake and processing. We describe here the use of FITC conjugated subtilisin in antigen uptake experiments and methods to track the processing of subtilisin both in vitro and in vivo in human cell lines.

Preparation and characterization of FITC-subtilisin
FITC-labelled subtilisin (FITC-subtilisin) was prepared by derivatizing amino groups on the PMSF-inactivated subtilisin, using 10-fold molar excess of FITC. FITC-subtilisin was purified and assayed for relative fluorescent units (RFU) per microgram protein. The RFU signal was linear from 0.1 to 100 RFU/well and conjugates ranged from 0.6 to 22 RFU/µg protein, corresponding to 0.06 to 2.1 moles of FITC per mole of protein. Conjugates with low ratios of FITC to subtilisin were obtained by increasing the concentration of enzyme in the reaction. Conjugates were TCAprecipitable and showed 89-93% of the fluorescein signal in the pellets. Generally 2 moles or less of FITC were coupled per mole of subtilisin.
FITC-subtilisin showed 10% enzyme activity compared to the unmodified subtilisin, as tested on the synthetic substrate, sucAAPF-pNA [20], as a result of residual PMSF bound to FITC-subtilisin (data not shown).

Identification of FITC-labelled sites on subtilisin using LC-MS
LC-MS was employed to confirm FITC modification of subtilisin. A deconvoluted LC-MS mass spectrometry profile of the modified protein showed free subtilisin (27,476 Da) and FITC-subtilisin with one (27,865 Da) and two moles of FITC (28, 255 Da) ( Figure 1). To localize the amino groups on subtilisin modified by FITC, TCA precipitated FITC-subtilisin was hydrolysed with trypsin, which cleaves preferentially at lysine residues. Figure 2 shows identification of subtilisin peptide 137-145 that contained the FITC-modification at the lysine 141 residue. Using fragmentation software analysis, the N-terminus and sites K256 and K141 were also identified as FITCmodification sites.

FITC-subtilisin is immunoreactive and degraded by human B-LCL extracts
To ensure that the overall structure of subtilisin was unchanged, we bound FITC-subtilisin with anti-subtilisin antibody and tracked immunoadsorption using the fluorescein label. Relative fluorescent units showed over 40% of the FITC-subtilisin bound to rabbit polyclonal antisubtilisin antibody (Table 1). We have routinely used this antibody to track unmodified subtilisin in western blots (data not shown). As a positive control, a mouse anti-FITC monoclonal antibody was used to immunoprecipitate the FITC-subtilisin. No significant binding was observed to normal rabbit gamma globulin control.
To determine whether FITC-subtilisin would be processed by cellular enzymes used in the antigen processing machinery, FITC-subtilisin was incubated with whole cell extracts from Preiss cells. To track processing, low molecular weight fragments were separated with a centrifugal filtration device and relative fluorescent units in the flowthrough were measured. Control samples contained PMSF to retard autolysis of subtilisin and prevent processing by the cellular enzymes. Cell extracts from Preiss cells generated significant amounts of low molecular weight peptides at pH 5.3, the pH of endocytic compartments within the cell, compared to controls (See Table 2). At pH 7, however, autolysis of FITC-subtilisin was comparable to the hydrolysis by the cell extracts. Similar results were obtained with whole cell extracts from cell line 8.1.6. These results showed that FITC-subtilisin was susceptible to hydrolysis in vitro by mammalian cellular enzymes and could be used to track and detect peptides generated from antigen processing.

Establishing optimal conditions for antigen uptake and processing in human B-LCL
Once we established that FITC-subtilisin was comparable in overall structure to unmodified subtilisin and capable of being processed in vitro, we tracked uptake and processing in vivo in cells. FITC-subtilisin was co-incubated with Preiss cells for 24 hours, the cells were lysed in acid and RFU were analyzed in the low molecular weight fractions of the cell lysates (See Table 3).
Using this relatively simple method, we tested several different conditions that could facilitate antigen uptake and processing. We compared uptake of enzymatically active FITC-subtilisin, TCA-precipitated FITC-subtilisin and autolyzed FITC-subtilisin in 8.1.6 cells (Table 4). Enzymatically active subtilisin degraded the cells and was not used in subsequent experiments. Significantly more TCAprecipitated subtilisin was associated with the washed cell pellet though only 0.06% RFU were recovered from the low molecular weight fraction. Similarly, incorporation of FITC-subtilisin in lysosomes and commercial carriers resulted in increased RFUs in the cell pellet but did not convert into significant increases in low molecular peptides (See Table 5). Multimers of FITC-subtilisin prepared by glutaraldehyde coupling or antibody FITC-subtilisin conjugates also failed to show increased processing. The early (2-12 hour) and late (36-48 hour) time points did not show significant increase in uptake and processing; neither did increasing antigen dose. Using greater numbers of cells as well as incubating antigen for 16 to 24 hours, however, did result in greater uptake and processing.

Identification of processed FITC-subtilisin peptides in human B-LCL cells
Based on our results of optimal conditions for uptake and processing, 4-6 × 10 9 cells were incubated with 0.2 mg/ml FITC-subtilisin (0.6 mole FITC/mole subtilisin) for 24 hours. The cell pellets were either acid treated directly as described above or processed for DR extraction; low LC/MS Analysis of FITC-Subtilisin molecular weight fractions were analyzed for RFUs and then by mass spectrometry. For DR-extraction, the cells were washed and lysed and loaded on a HLA-DR immunoaffinity column. The column flow-through had significant RFU. This was fractionated and the low molecular weight fraction was concentrated. HLA-DR molecules purified by the immunoaffinity column were isolated and DR-bound peptides were acid-eluted from these molecules. Half as many RFUs were detected from the DReluted peptides (See Table 5). Both samples were analyzed by LC-MS spectroscopy. A subtilisin peptide was identified from the flow-through sample.
Subtilisin peptide 114-126 found in the flow-through was confirmed by spiking a synthetic version of this pep-tide in control cell extracts and comparison of the synthetic peptide with the unknown peptide by retention time, m/z charge, and fragmentation pattern. Overall, three subtilisin regions were identified from cellular extracts of cells incubated with antigen but not from control cell extracts not incubated with antigen, by ms/ms fragmentation in multiple experiments (See Table 6). Nested sets of peptides from these regions were consistently observed in two or more experiments. However, peptides were not detected in the DR-eluted fraction of the extracts.

MS/MS A A V D K V A S A
Base Peak Search at m/2 = 390.0 Da    TCA-precipitated FITC-subtilisin Uptake, no increase in processing 3.
FITC-subtilisin antibody conjugate Uptake, no increase in processing 5.
FITC-subtilisin crosslinked with glutaraldehyde Uptake, no increase in processing 6.
Antigen dose 100-900 µg/mL 100-200 µg/mL best tilisin and HLA-DR was extracted from the cell lysates. These cells are more proficient at antigen uptake than B cell lines though less amenable to large-scale cultures. DRbound peptides were eluted and analyzed on the Q-TOF and a subtilisin peptide was identified (Table 6) in a single experiment. This peptide corresponded to a region of subtilisin, 70-84 that had been identified as an epitope in another study (unpublished data).

Discussion
The use of fluorescent conjugates to investigate immunological processes has a long history. This study exemplifies their use in tracking antigen to identify optimal conditions for antigen uptake and processing.
Subtilisin is one of the most extensively studied of all bacterial proteins, due to its commercial importance [20,21]. Our laboratory is interested in its potential role as an antigen [13]. In order to identify naturally processed peptides from subtilisin, we devised a simple way to label, stabilize, measure, and track the protein in cell studies. We demonstrate the preparation and characterization of FITC-subtilisin conjugates, and analysis of antigen taken up and processed by human cell lines.
We have shown that subtilisin can be labelled with FITC with generally two or fewer moles of FITC per mole of protein. These conjugates retain antigenicity and up to 30% of their enzymatic activity on small substrates. These conjugates can be degraded by cellular extracts of mam-malian cells at pH 5.3 showing the FITC label does not prevent hydrolysis. We show that the fluorescein residues on FITC-subtilisin are present on the N terminus and two other lysine residues. Though the final processed peptides may contain these fluorescein residues, they are present in minute amounts in the low molecular weight fractions, precluding RFU detection within the sensitivity of our current detection systems.
Mass spectrometry analysis of the low molecular weight fraction showed detectable and identifiable subtilisin peptides when the specific RFU/µl of sample was 0.5 or more. Thus, using the 10 K fraction, if 50 RFU are isolated after peptide elution, assuming an average mw of 1500 daltons, this signa1 represents about 55 ng of peptides. The 55 ng peptides when prepared for mass spectrometry analysis would be in the picomole to subpicomole range, which is consistent with current mass spectrometry detection limits and with the calibrated sensitivity of our LC-MS system at 0.5-1.0 picomoles of peptide spiked into cell extracts without antigen. Our detection limits are also comparable to reports in the literature where self-peptides presented by class II MHC molecules induced by a measles vaccine virus infection were present at 0.5 pmol and identified [22].
Our results show that although cell associated RFUs following extensive washes could be indicative of impressive uptake responses; these did not translate to processing of antigen into low molecule weight peptides in the cell  extracts or into peptides bound to MHC molecules. We believe the high RFUs associated with cells may be nonspecific adherence of the antigen to the cell membranes. The B-LCL cells endocytosed very minute amounts of antigen (less than 1% of incubated antigen), of which only 10-20% were low molecular weight peptides of less than 10,000 daltons. In conclusion, despite testing several different conditions, none increased antigen uptake significantly in the B-LCL cells, consistent with the low efficiency of non-receptor mediated antigen uptake by B cells [9,23].
Our peptide analysis results from multiple experiments shows three distinctive regions of the subtilisin molecule that are detected in the cellular extracts by mass spectrometry. The one DR-bound peptide detected is from a different region of the molecule and corresponds to a previously identified subtilisin epitope (unpublished data). This DR-bound peptide was analyzed on the Q-TOF, which is more sensitive than the LC-MS system. The peptides detected in the cellular extracts by LC-MS could be peptides that are processing intermediates of peptides to be bound and presented on class I or class II molecules or simply regions of the protein more stable in the presence of cellular degrading enzymes. Our data do not distinguish between these possibilities.

Conclusions
In conclusion, we have demonstrated that FITC conjugation can be used successfully to track antigen uptake and processing and detect processed peptides in the cellular extracts and DR-bound peptides in human cells. This method is simple, efficient, and relatively inexpensive. Thus it would be useful to study the uptake and processing of several other antigens. Improved mass spectrometry methods to detect the presence of MHC bound naturally processed peptides would further enhance the utility of the described method. Our future studies will focus on studying uptake and processing of other FITC labelled antigens and improving mass spectrometry methods to detect the presence of other MHC bound naturally processed peptides.

Enzyme
Subtilisin was a single amino acid mutant (BPN' Y217L) prepared from Bacillus amyloliquefaciens (Swissprot accession number P00782) and purified by crystallization. It was formulated in 50% glycerol at 55 mg/ml and stored at -17°C, where it was stable. Subtilisin activity was measured in a kinetic assay with the synthetic substrate sucAAPF-pNA as previously described [20].

Pulsing and harvesting cells
Cells were cultured to 1-1.2 × 10 6 /ml and resuspended in 400 ml of complete medium to a concentration between 0.5-1 × 10 7 per ml and 150-250 µg/mL PMSF inactivated FITC-subtilisin. Cells were cultured at 37°C for 2 hr and then diluted 1:2 in complete medium. After 20 hrs cells were washed extensively in Dulbecco's PBS.